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Abstract:

A nicking endonuclease is described which has an amino acid sequence with
at least 70% identity to SEQ ID NO:6 and comprising a mutation at least
one of an arginine or gutamic acid corresponding to position 507 and
position 546 respectively in SEQ ID NO:6.

Claims:

1. A nicking endonuclease, comprising: an amino acid sequence with at
least 70% identity to SEQ ID NO:6 and comprising a mutation at an
arginine corresponding to position 507 in SEQ ID NO:6.

2. A nicking endonuclease, comprising; an amino acid sequence with at
least 70% identity to SEQ ID NO:6 and comprising a mutation at a glutamic
acid corresponding to position 546 in SEQ ID NO:6.

3. A nicking endonuclease according to claim 1, further comprising a
mutation at a glutamic acid corresponding to position 546 in SEQ ID NO:6.

4. A nicking endonuclease according to claim 1 or 2, wherein the gene
encoding the endonuclease has at least 90% sequence identity with SEQ ID
NO:5.

5. A nicking endonuclease according to claim 1 or 3, wherein the arginine
is changed to an aspartic acid and the glutamic acid is changed to a
valine.

6. A method of forming a nicking endonuclease from a restriction
endonuclease having an amino acid sequence, comprising: mutagenizing at
least one of an arginine or a glutamic acid in the amino acid sequence by
targeted mutagenesis; cloning the mutagenized restriction endonuclease
and assaying the mutant for nicking activity.

7. A method according to claim 7, wherein the restriction endonuclease is
BsmI or an isoschizomer or neoisoschizomer thereof.

[0004]Type II restriction endonucleases (REase) generally have two
subunits forming either a homodimer or a heterodimer.

[0005]For homodimeric EcoRV, Stahl et al. (Proc Natl Acad Sci USA
93(12):6175-80 (1996)) described combining a subunit with an inactive
catalytic activity with a second subunit with a deficiency in DNA binding
to produce a nicking endonuclease that is non-specific with respect to
which strand is nicked.

[0007]Xu (Proc. Natl. Acad. Sci. USA 98:12990-12995 (2001)) reported the
creation of N.AlwI by domain exchange between the Type IIS REase AlwI and
a homologous, naturally occurring nicking enzyme, N.BstNBI. This nicking
endonuclease predominantly nicks the top DNA strand of a DNA duplex as a
monomer. This domain exchange method requires prior knowledge of the
dimerization domain and a relatively high amino acid sequence similarity
with a naturally existing nicking enzyme.

[0008]Site-directed mutagenesis of MlyI REase resulted in variants in
which the dimerization function was disrupted. The resulting nicking
enzyme is strand-specific, cleaving the top strand of the wild type
recognition sequence. However, no bottom strand nicking enzyme was ever
isolated from MlyI (Besnier C. E. et al. EMBL Rep. 2:782-786 (2001).

[0009]The DNA nicking activity of BfiI can be enhanced by alteration of
reaction conditions. By lowering the pH value in the cleavage reaction,
the BfiI REase can be converted to a bottom-strand specific nicking
enzyme (Sasnauskas G. et al. Proc. Natl. Acad. Sci. USA 100:6410-6415
(2003)).

[0010]Zhu (J. Mol. Biol. 337:573-583 (2004)) used random mutagenesis and
back-crosses with the Type IIS restriction endonucleases to generate
BsaI, BsmAI and BsmBI nicking variants. There was no selectivity in
nicking strand specificity. The random mutagenesis method required
screening a large number of mutants.

[0011]Samuelson (Nucl. Acids Res. 32:3661-3671 (2004)) designed a SapI
substrate site into the expression plasmid to allow for in vitro
selection of plasmid clones from a randomly mutated SapI expression
library possessing a site-specific and strand-specific nick.
Bottom-strand nicking enzymes yielded Nb.SapI-1 containing a critical
R420I substitution near the end of the protein while a separate
top-strand selection procedure yielded several SapI variants with a
distinct preference for top-strand cleavage.

[0012]Nicking endonucleases have been created from heterodimeric Type IIT
including Bpu10I (Stankevicius K. et al. Nucl. Acids Res. 26:1084-1091
(1998), EP 1176204 A1, July 2000, BbvCI (US patent application
2003/0100094 and BslI (Hsieh et al. J. Bacteriol. 182:949-955 (2000))
These nicking endonucleases were formed by inactivation of the catalytic
activity of one subunit in the heterodimer.

[0013]Nicking BsmAI and BsmB1 have been made by error prone PCR and site
directed mutagenesis (U.S. application Ser. No. 11/013,235).

SUMMARY OF THE INVENTION

[0014]In an embodiment of the invention, a nicking endonuclease is
described that has an amino acid sequence with at least 70% identity to
SEQ ID NO:6 and includes a mutation at an arginine corresponding to
position 507 in SEQ ID NO:6. The nicking endonuclease may further contain
a mutation at a glutamic acid corresponding to position 546 in SEQ ID
NO:6. The DNA encoding the nicking endonuclease has at least 90% sequence
identity with SEQ ID NO:5.

[0015]In an embodiment of the invention a nicking endonuclease is
described that has an amino acid sequence with at least 70% identity to
SEQ ID NO:6 and includes a mutation at a glutamic acid corresponding to
position 546 in SEQ ID NO:6. The DNA encoding the nicking endonuclease
has at least 90% sequence identity with SEQ ID NO:5.

[0016]In an example of the above, the arginine can be changed to an
aspartic acid and the glutamic acid to a valine.

[0017]In an embodiment of the invention, a method is provided of forming a
nicking endonuclease from a restriction endonuclease having an amino acid
sequence. This method includes: mutagenizing at least one of an arginine
or a glutamic acid in the amino acid sequence by targeted mutagenesis;
cloning the mutagenized restriction endonuclease and assaying the mutant
for nicking activity.

[0018]The method is exemplified by starting with BsmI restriction
endonuclease or an isoschizomer or neoisoschizomer thereof and
mutagenizing this enzyme in the manner described above.

[0024]FIG. 6 shows the DNA and protein sequence for BsmI restriction
endonuclease. The arginine codons and residues are highlighted in bold
font and the glutamic acid codons and residues are highlighted in
italics.

[0026]Methylase protected cells are generated by cloning the appropriate
methylase gene into a vector for example pACYC184, capable of being
replicated in a host bacterial cell such as E. Col. The restriction
endonuclease gene of interest is cloned in a second vector, for example,
pUC19. The restriction enzyme gene can alternatively be expressed in
other vectors with T7 promoter, phage SP6 promoter, Ptrp, Ptac,
lacUV5 promoter, PL, PR, or Para and used as the
template for inverse PCR.

[0027]Once cloned, the restriction endonuclease gene can be mutated using
any method of targeted mutagenesis known in the art. For example, inverse
PCR is used in Example 1. It was noted previously that a nicking
endonuclease, which was created by random mutagenesis of the BsaI
restriction endonuclease gene had mutated arginine residues.
Consequently, present embodiments of the invention target arginine
residues. Each Arg codon can be identified from the gene sequence and
then changed one at a time to a different codon, for example, aspartic
acid. The amino acid encoded by the altered codon can be any non-acidic
amino acid. The choice of aspartic acid was arbitrary. Individual mutants
can be cloned and assayed for nicking activity and desirably an absence
of double strand cleavage activity. In addition to arginine residues,
other targets of mutation include Asp, Gln, and Lys, which may be mutated
to amino acids having a different charge. Once a suitable nicking
endonuclease is obtained, the enzyme can be purified and assayed for
nicking activity (US 2003-0100094 A1) and strand nicking specificity (Xu
et al. 2004 J. Mol Biol. 337:573-583).

[0028]In one embodiment of the invention, bsmIR is mutated to yield a
nicking endonuclease. This enzyme has 30 Arg residues although the number
of Arg residues varies among different endonucleases. By substituting
each arginine for an aspartate in thirty separate clones (see below), one
nicking endonuclease (isolate #26) with high level of nicking activity
and substantially no double strand cleavage activity was obtained. This
mutant was identified as R507D. When the allele of isolate #26 was
sequenced, it was found to carry two additional mutations/amino acid
substitutions and was characterized as R507D/G509V/E546V. The nicked
pBR322 DNA was subjected to run-off sequencing to determine the strand
specificity. The triple mutant R507D/G509V/E546V nicked the bottom strand
of BsmI site with the strand specificity of G'CATTC. An additional
nicking endonuclease was prepared which was derived from #26 but
contained a single mutated Glutamic acid (E546V). This mutant had some
minor double strand cleavage activity of less than 20% (FIG. 4).

[0029]Mutations identified as effective for converting a restriction
endonuclease into a nicking endonuclease can be introduced into
isoschizomers to generate strand specific nicking variants. For example,
for BsmI, the R507D and/or E546V substitutions (or R507X, E546X, X=the
rest of 19 amino acid residues) can be introduced into BsmI
isoschizomers/neoschizomers such as BsaMI, Mva1269I, PctI, Asp26HI,
Asp27I, Asp35I, Asp36I, Asp40I, Asp50I, BscCI, Uba1382I, and Uba1415I in
the equivalent positions to generate strand-specific nicking variants.

[0030]The present embodiments are further illustrated by the following
Examples. These Examples are not intended to be limiting.

[0031]The references cited above and below as well as provisional
application Ser. No. 60/590,441 are hereby incorporated by reference.

EXAMPLES

Example 1

Construction of a High Expression Clone of BsmI Endonuclease by Targeted
Mutagenesis

[0032](a) Cloning bsmIM into pACYC184

[0033]The bsmIM gene was first amplified by PCR and cloned into pACYC184
to construct a protected expression host ER2683 [pACYC-bsmIM]. ER2683
carries the lacIq gene and therefore Lac repressors are
over-produced in this strain.

(b) Cloning bsmIR into pUC19

[0034]The bsmIR gene was amplified in PCR using the following primers:

PCR was conducted as follows: 4 units of Deep Vent DNA polymerase, 2, 4,
and 8 mM MgSO4, 1× Thermopol buffer, 0.4 nM dNTP, 1 μg of
Bacillus stearothermophilus NUB36 genomic DNA template. 0.24 μg
(˜0.4 to 0.8 mM) primers 303-095 and 303-096. The PCR DNA was
digested with SphI and ZraI and ligated to pUC19 with compatible cohesive
ends (SphI/SfoI). Cell extracts from 8 clones displayed BsmI endonuclease
activity. The entire bsmIR gene for the #2 clone was sequenced and was
found to encode the wild-type sequence (see FIG. 6). This plasmid was
named pUC-bsmIR and used for expression and mutagenesis. The expression
strain was ER2683 [pACYC-bsmIM, pUC-bsmIR]. The BsmI restriction
endonuclease yield was estimated to be at approximately 106
units/gram of wet cells.

Example 2

Mutagenesis Scanning of BsmI Endonuclease to Isolate Nicking Variants

[0035]Using inverse PCR, each Arg codon was changed to Asp codon (GAT)
with one Arg mutation per clone. A total of 60 PCR primers were
synthesized in order to make the 30 site-directed mutants. The primers
were about 39 nucleotides in length which provided sequence on either
side of the arginine codon (see FIG. 6). For example, in order to
mutagenize Arg507 to Asp507 (R507D), the following primers were made:

[0067]In summary, BsmI variant #26 (R507D) displayed high DNA nicking
activity and substantially no ds-DNA cleavage activity. Variants R123D,
R159D, R364D, R367D, and R526D displayed low to intermediate DNA nicking
activity. The rest of the mutants displayed ds-DNA cleavage activity. The
most active nicking variant #26 was further characterized. The whole gene
was re-sequenced using 6 primers. It was found that in addition to the
expected mutation (R507D), there are two additional mutations in the
allele. One amino acid substitution (G509V) was introduced by the
mutagenic primer. The third mutation/amino acid substitution (E546V) was
introduced during inverse PCR. It's known that mutations can be
introduced during PCR (the error rate of Deep Vent DNA polymerase is
2×10-5 per replicated nucleotide). Thus, BsmI nicking variant
#26 carried three amino acid substitutions: R507D/G509V/E546V. The DNA
nicking activity of Nb.BsmI (R507D/G509V/E546V) is shown in FIGS. 1, 2
and 3.

[0068]To investigate the importance of E546V substitution in contributing
to the nicking phenotype, the E546V mutation was separated from
R507D/G509V by restriction fragment exchange with the wild-type (wt)
coding sequence. Cell extract of E546V variant was prepared by sonication
and the nicking activity was assayed on pBR322 supercoiled DNA. It was
found that the single mutant E546V displayed higher nicking activity than
the triple mutant Nb.BsmI (R507D/G509V/E546V), but the ds-DNA cleavage
activity is increased somewhat (to about 10%). Fortuitously, when amino
acid substitution E546V was combined with R507D substitution (G509V is
not likely an important change), the ds-DNA cleavage activity was
minimized. It is therefore concluded that some synergistic effect between
the two mutations gives rise to the desired nicking endonuclease
activity. The nicking variants E546V and R507D/G509V/E546V were partially
purified by chromatography through heparin Sepharose and DEAE Sepharose
columns and used to nick pBR322 supercoiled DNA. The nicked DNA was
gel-purified and used for run-off sequencing. It was determined that
nicking variants E546V and R507D/G509V/E546V were bottom-strand nicking
enzymes with the specificity of G CATTC Strandedness was determined by
the techniques described in Xu et al. Journal of Molecular Biology
337:573-583 (2003)). ( indicating the nicking position.) The DNA nicking
activity of E546V variant is shown in FIG. 4.

Example 3

Purification of Nicking Enzyme Nb.BsmI (R507D/G509V/E546V)

[0069]Nb.BsmI (R507D/G509V/E546V) was purified by chromatography through
Heparin Hyper-D, Heparin-TSK, Source Q, and Superdex 75 columns or by
chromatography through other cation or anion exchange columns or
molecular weight sizing column.

[0070]Two hundred and sixty grams of frozen cell pellet were resuspended
in 780 ml of buffer A (20 mM KPO4 pH 7.1, 0.1 mM EDTA, 10 mM
β-mercaptoethanol, 0.2 M NaCl, 5% glycerol). The cells were broken
in the Gaulin press and cell debris were removed by high-speed
centrifugation. The pH after breakage was 8.0. The assay for determining
the nicking activity was done using 322 pBR as the substrate and looking
for the supercoiled DNA to be nicked into the relaxed circular form.

1) The first column in the purification was a large Heparin Hyper-D
column. This was a capture step. The supernatant was loaded onto the
column and a gradient of 0.1 M NaCl to 2.0 M NaCl was applied. The enzyme
eluted at about 1.0 M NaCl. The active fractions were pooled and diluted
with Standard Buffer, SB (20 mM KPO4 pH 7.1, 0.1 mM EDTA, 10 mM
β-mercaptoethanol, 5% glycerol) and salt adjusted to a final
concentration of 0.2 M NaCl. This was loaded onto the next column.2) The
second column was a 40 ml Heparin-TSK column. This column concentrated
the enzyme. A salt gradient was run using SB/0.2 M NaCl to 2.0 M NaCl. A
much sharper peak was obtained and pooled. This was dialyzed against 4 L
of SB/0.1 M NaCl. After this purification step, the enzyme was fairly
concentrated so a Source-Q was run to eliminate the DNA.3) A 60 ml Source
Q column was loaded with the dialyzed enzyme equilibrated to SB/0.1 M
NaCl. As expected, most of the contaminant DNA bound to the Source Q
resin. The flow-through was collected and a gradient was run over the
Source Q to see if any of the enzyme had bound, but all of the enzyme had
come out in the flow-through.4) The enzyme in the Source Q flow-through
(at a NaCl concentratration of 0.1 M) was loaded back onto the Heparin
TSK column and a sharp gradient was applied up to 2.0 M NaCl. The enzyme
was pooled to keep the volume down to 30 ml and loaded onto a molecular
weight sizing column.5) The Superdex 75 was run with 4 L of 20 mM
Tris-HCl, pH 7.6, 0.1 mM EDTA, 10 mM b-mercaptoethanol, 0.5 M NaCl, 5%
glycerol. Three symmetric peaks were detected in the fraction collector
and the middle peak had most of the nicking activity. The fractions in
the middle peak were pooled and dialyzed into storage buffer (15 mM
Tris-HCl pH 7.2, 0.1 mM EDTA, 1 mM DTT, 0.15 M NaCl, 50% glycerol).6) The
final enzyme was titered for concentration and subsequent quality
controls were done. The purified Nb.BsmI (R507D/G509V/E546V) is shown in
FIG. 5.

[0072]The nicking assays were also performed at temperatures ranging from
25° C. to 75° C. in NEB buffer 3 (100 mM NaCl) (New England
Biolabs, Inc., Ipswich, Mass.). FIG. 2 shows that Nb.BsmI is active in
the wide range of temperatures. It is active at 25° C. to
75° C. in the nicking reactions.

[0073]In the DNA nicking reactions, it is desirable to minimize the ds-DNA
cleavage. Therefore, 4- to 80-fold over-digestions were performed on
pBR322 in order to detect nicked circular DNA and/or linear DNA. The
following assay condition was used: 0.5 μg pBR322 substrate, 5 μl
10× buffer 3, 1-5 μl diluted and undiluted Nb.BsmI (1 to 40
units), adjusted volume to 50 μl with sterile distilled water. Nicking
reactions were carried out at 65° C. for 1 h. FIG. 3 shows that 40
units of Nb.BsmI generated a weak linear band (at ˜80-fold
over-digestion). No linear DNA was detected with 1 to 30 units of Nb.BsmI
digestion. It was concluded that no more than 60-fold over-digestion
should be carried out in order to minimize ds-DNA cleavage.

[0074]The E546V substitution was introduced during inverse PCR
amplification, which resulted in a nicking phenotype. Site-directed
mutagenesis can change each Asp residue to a non-charged hydrophobic
residue such as Val, Met, Ile, Leu, or any other amino acid residues
other than Asp and Glu. Cell extracts can be prepared from the mutant
cell cultures and assayed for DNA nicking activity on appropriate DNA
substrates. The nicking strand specificity can be determined by run-off
sequencing.

[0075]Alternatively, the Asn, Gln, and Lys residues in BsmI restriction
endonuclease can be mutated to generate nicking variants either by
site-directed mutagenesis, localized random mutagenesis or random
mutagenesis. The nicking variants can be purified by cation or anion
exchange columns or molecular weight sizing column. BsmI nicking variants
can also be purified by heat denaturation. E. coli host proteins can be
heat-denatured by heating the cell extracts at 50° C. to
75° C. for 20 to 60 min. Heat-denatured proteins can be removed by
high speed centrifugation. The supernatant contains the partially
purified nicking enzyme.